Improving Immune Checkpoint Blockade Efficacy Through the Design and Optimization of 5'-Triphosphate RNA Loaded Nanoparticles
Jacobson, Max Edward
Here, I develop and then further optimize 3pRNA-NPs: a nanoparticle delivery platform capable of activating the innate immune system through engagement of the cytosolic pattern recognition receptor RIG-I. Both targeting cytosolic receptors as well as utilizing RNA therapeutics are met with historic barriers to efficacy, including RNA degradation, poor cellular uptake, endosomolytic recycling, and lysosomal degradation. 3pRNA-NPs utilize pH responsive, membrane destabilizing polymers electrostatically complexed to the RIG-I agonist 5'ppp dsRNA, to overcome these barriers, drastically increasing the magnitude of the innate immune response. First, we use an already established pH-responsive membrane destabilizing polymer to formulate 3pRNA-NPs. We find that these particles are capable of inducing an immunostimulatory response, including the production of immune activating cytokines and T-cell recruiting chemokines in both murine colon cancer cells in addition to murine myeloid cells that comprise a typical tumor microenvironment. Furthermore, we found that 3pRNA-NP treatment also caused murine cancer cell death. We administer a combinatorial therapy comprising these 3pRNA-NPs in conjunction with anti-PD-1 in a murine colon cancer model and find that the combinatorial treatment resulted in redcued tumor burden as well as 3 tumor-free mice that resisted tumor rechallenge. In order to further optimize 3pRNA-NPs, we synthesize a series of pH-responsive polymers for formulations. We establish a relationship between polymer composition and pH-responsive properties across two parameters: hydrophobic monomer side chain length and second block composition. Next, we evaluate the ability of these series polymers to enhance the immune response of 5'ppp dsRNA, and further investigate four lead polymers capable of vastly enhancing 5'ppp dsRNA treatment. Although these polymers behave similarly, we find that NPs comprising 5'ppp dsRNA complexed with the series polymer “P-b-DA450” demonstrate enhanced stability and immunostimulatory response in murine breast tumors. While 3pRNA-NP dose and treatment regimen remain to be optimized for maximum therapeutic benefit, these studies demonstrate the importance of carrier design in immunotherapeutic targeting and set the stage for future investigation into the development of new delivery technologies for this promising class of innate immune agonist.